Homeodomain (HD) proteins comprise a large family of transcription factors (TFs) that function in a variety of ways to direct development, often acting in the same cells but with each one exerting a different regulatory effect. These activities include the specification of regional, tissue and single cellular fates. HD proteins act by binding to related DNA sequences within transcriptional enhancers where they either activate or repress the expression of the associated gene. While some subclasses of HDs are known to bind unique DNA sequences, many HD proteins recognize highly similar motifs characterized by a canonical TAAT core, thereby raising the question of how HD response specificity is generated in different developmental contexts. To address this question, in collaboration with Dr. Martha Bulyks laboratory (Brigham and Women's Hospital), we previously used a high-throughput, protein binding microarray (PBM) assay to determine the complete spectrum of DNA binding specificities of a large number of HDs that are expressed in the Drosophila embryonic mesoderm. These studies revealed that many HDs recognize identical or highly similar sequences, but with each HD protein also preferentially binding to variant sequences that are not bound by related HDs. A series of mutagenesis experiments involving cell-specific enhancers established that these preferred HD binding motifs indeed account for the transcriptional response specificity of a HD class that determines muscle founder cell identities. Furthermore, computational analyses revealed that such HD-preferred binding sites are over-represented in putative enhancers associated with genes that are responsive to the corresponding HD TF, a result which supports the generality of this previously unrecognized mode of transcriptional regulation by this developmentally important class of TFs. Since multiple HD family members recognize the same DNA sequences in cell- and tissue-specific enhancers, it is difficult to ascertain the role of a particular HD subclass in regulating that element and thereby mediating specific developmental functions. To address this question, we took advantage of earlier studies in the Drosophila embryonic mesoderm where HD TFs were shown to be required not only for establishing segmental identities (the Hox subclass of HDs), but also for mediating tissue and individual cell fate specification responses (the NK-2, Six and cellular identity or I-HD subfamilies). In these experiments, we applied our prior knowledge of the complete range of DNA binding specificities of a set of mesodermal HD proteins determined by PBM assay to dissect the separate contributions of different HD subclasses to the regulation of mesodermal enhancers that control the expression of both upstream (transcription factor) and downstream (differentitation effector) components of mesodermal cell regulatory networks. The experimental design involved modifying the nucleotide sequences of four well-characterized mesodermal enhancers such that they are recognized by either no HDs or by a by single subclass of this family of TFs. The activities of these mutagenized enhancers were then determined in a series of transgenic reporter assays. These studies revealed that individual mesodermal enhancers receive separate transcriptional input from both IHD and Hox subclasses of HDs. In addition, we demonstrated that enhancers regulating upstream components of the mesodermal regulatory network are targeted by the Six class of HDs. Finally, we established the necessity of NK-2 HD binding sequences to activate gene expression in multiple mesodermal tissues, supporting a potential role for the NK-2 HD TF Tinman (Tin) as a pioneer factor that cooperates with other factors to regulate cell-specific gene expression programs. Collectively, these results underscore the critical role played by HDs of multiple subclasses in inducing the unique genetic programs of individual mesodermal cells, and in coordinating the gene regulatory networks directing mesoderm development. In a related set of experiments, we are collaborating with Dr. Kevin Whites laboratory (University of Chicago) to characterize the transcriptional programs regulated by HD TFs and their cofactors in the Drosophila embryonic mesoderm by defining the in vivo binding profiles of a subset of these proteins in flow sorted mesodermal cells using chromatin immunoprecipitation combined with high-throughput DNA sequencing (ChIP-seq). Findings from these investigations are expected to extend and complement those already completed, as summarized above.